Abstract Harnessing the therapeutic promise of immunotherapy against cancer has evolved from strategies focused on the antigen-recognition mechanisms of adaptive immunity (e.g. checkpoint inhibition and CAR-T cells) to strategies leveraging direct cytotoxicity mechanisms of innate immunity. The clinical impact of these exciting new immunotherapy strategies can be realized only with customized technologies and products for Natural Killer (NK) cells, central to direct cytotoxicity mechanisms of innate immunity - e.g. achieving reliable, low-risk and low-cost ?ex vivo expansion of patient-derived NK cells, which remains a strong unmet need and an underserved niche in immunotherapy. Our proposal aims to achieve a serum-free media supplement for NK cells through a novel combination of plant-derived ingredients and traditional media supplements, guided in prototyping with a fractional-factorial statistical toolkit for recipe optimization. To achieve this goal, our Phase-I proposal outlines two specific aims - one to optimize the prototype serum-free media recipe with cell-proliferation and viability performance being key end-points, and two to further fine-tune the prototype serum-free media recipe with cytotoxicity and biomarker end-points pertaining to both apoptosis and necrosis mechanisms of direct cytotoxicity by NK cells, both specific aims guided with a statistical approach for efficient optimization. For the pursuit of both these specific aims, the proposed NK cell line is the p53 mutation based KHYG1 NK cell line, followed by a confirmation of the key experimental end-points with 3 distinct batches of primary human NK cells. At the end of Phase-I, we envision a lead formulation for a novel and effective serum-free media supplement that is specific to NK cell growth and maintenance of NK cell phenotype and functions (e.g. cytotoxicity mechanisms). The Enable Life Sciences LLC team is well positioned to pursue the project goal starting with the proposed Phase-I specific aims. In addition to being led by the proposed Principal Investigator (PI), Dr. Rachit Ohri (CEO and Founder of Enable Life Sciences) who has the relevant expertise, the proposed effort will be well supported by other team members such as Donna Sonntag (MS from the University of Massachusetts, Lowell) and other scientists and support staff. The Enable Life Sciences labs in Worcester MA already houses the requisite equipment and instrumentation, namely flow cytometers, Luminex, microscopes, BL2 (biosafety level 2) biological safety cabinets etc. Moreover, Enable Life Sciences is well entrenched in an ecosystem in an around Worcester MA, which is deeply facilitative for the proposed objectives of this Phase-I proposal. These include a supportive R&D and business environment, for example access to advanced instrumentation such as multi-laser flow cytometry, confocal fluorescence microscopy etc. at University Core Research Facilities (CRFs) at numerous campus sites in the North-East. In a nutshell, our proposal includes carefully crystallized, relevant and achievable objectives and methodologies, additionally we are well-positioned as an organization to achieve the envisioned serum-free media supplement for NK cells.